Diuretic effects and urinary electrolyte excretion induced by Aspidosperma subincanum Mart. and the involvement of prostaglandins in such effects

Diuretic effects and urinary electrolyte excretion induced by Aspidosperma subincanum Mart. and the involvement of prostaglandins in such effects

Journal of Ethnopharmacology 163 (2015) 142–148 Contents lists available at ScienceDirect Journal of Ethnopharmacology journal homepage: www.elsevie...

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Journal of Ethnopharmacology 163 (2015) 142–148

Contents lists available at ScienceDirect

Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep

Research Paper

Diuretic effects and urinary electrolyte excretion induced by Aspidosperma subincanum Mart. and the involvement of prostaglandins in such effects Emmeline Flor Ribeiro a, Carolina de Fátima Reis a, Flávio Silva de Carvalho b, João Pedro Silva Abreu b, Andréa Fernandes Arruda b, Clévia Ferreira Duarte Garrote a, Matheus Lavorenti Rocha a,n a b

Faculty of Pharmacy, Federal University of Goias, Avenida Universitária s/n, 74605-220 Goiânia, GO, Brazil Institute of Chemistry, Federal University of Goias, Campus Samambaia, Caixa Postal 131, 74001-970 Goiânia, GO, Brazil

art ic l e i nf o

a b s t r a c t

Article history: Received 29 October 2014 Received in revised form 14 January 2015 Accepted 19 January 2015 Available online 24 January 2015

Ethnopharmacological relevance: Aspidosperma subincanum Mart. is a medicinal herb known for its diuretic properties and used for the treatment of cardiovascular-related illnesses. Although our earlier study has shown that the ethanol extract of Aspidosperma subincanum (EEAS) induces hypotension and vasodilation, no scientific data have been recorded to evaluate the diuretic effects of this Brazilian medicinal plant. The aim of this study was to evaluate the diuretic activity of EEAS, and possible mechanism of action, using Wistar rats. Material and methods: EEAS (60 and 120 mg/kg), furosemide (20 mg/kg) or saline (control) were orally administered to rats individually held in metabolic cages for urine collection 1, 2, 4, 6, 8, 12 and 24 h after treatment. In order to evaluate the involvement of prostaglandins in the diuretic action of EEAS, the animals received piroxicam (5 mg/kg i.p.), a nonselective inhibitor of cyclooxygenase, before treatment with EEAS at 120 mg/kg. The control groups received only saline (NaCl, 0.9%), or saline and piroxicam. Urinary volume, electrolyte excretion and pH were measured. Results: Oral administration of EEAS 60 and 120 mg/kg significantly increased diuresis and electrolyte excretion of Na þ and K þ on a continuous basis throughout the study period. Both EEAS 60 and 120 mg/ kg caused a relative increase of around 77% and 142%, respectively, in cumulative diuresis compared with the control group. From 4th hour until the end of the experiment, the group treated with EEAS 120 mg/ kg provided a greater excretion of Na þ than the furosemide group. The diuretic effects of EEAS were neutralized by piroxicam between 4 and 8 h after treatment. Conclusion: The results suggest that EEAS could present compound(s) responsible for diuretic activities, and the mechanism could involve the prostaglandin system. & 2015 Elsevier Ireland Ltd. All rights reserved.

Keywords: Aspidosperma subincanum Diuretic Hypertension Electrolytes Prostaglandins

1. Introduction Cardiovascular diseases constitute the leading cause of disability and death worldwide (Rapeport and Middlemost, 2012). Conditions such as hypertension lead to other types of disorder, such as strokes, kidney and heart diseases, and hence need to be treated (Gupta et al., 2010). Common clinical strategies for reducing blood pressure include using different classes of drugs among which diuretics are highlighted (Gupta and Neyses, 2005; McManus and n Correspondence to: UFG, Faculty of Pharmacy, Av. Universitária com 1 Avenida s/n, Setor Universitário CEP, 74605-220 Goiânia, GO, Brazil. Tel.: þ 55 62 3209 6440; fax: þ55 62 3209 6037. E-mail address: [email protected] (M.L. Rocha).

http://dx.doi.org/10.1016/j.jep.2015.01.023 0378-8741/& 2015 Elsevier Ireland Ltd. All rights reserved.

Caulfield, 2012 McManus et al., 2012). Diuretic drugs increase the rate of urine flow and adjust the volume and composition of body fluids. Drug-induced diuresis is beneficial for the treatment of various disorders such as nephritis, chronic renal failure, hypertension, eclampsia and heart failure (Gupta and Neyses, 2005; Wile, 2012). Despite their high efficiency, many diuretics have been associated with undesirable adverse effects, including electrolyte imbalance, metabolic alterations, activation of the renin– angiotensin and neuroendocrine systems, and impairment of the sexual function (Ellison and Loffing, 2009; Wile, 2012). Against this background, the search for new agents with diuretic properties is of major interest to public health. Down the years medicinal plants have been a highly esteemed source of chemical substances with potential therapeutic effects.

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In recent times the Brazilian Ministry of Health launched two major programs involving research and the use of medicinal plants: the National Program for Medicinal Plants and Herbal Medicines (Ministério da Saúde et al., 2009) and the National Policy on Integrative and Complementary Practices (Ministério da Saúde et al., 2006). Among the interesting species found in folk medicine, the Aspidosperma genus comprises approximately 260 species which can be found from Mexico to Argentina (Jacomé et al., 2004). Aspidosperma species are extensively used throughout Latin America for medicinal purposes (Barbosa et al., 2003; Oliveira et al., 2009). Among the alkaloids found in genus Aspidosperma with pharmacological activities have been identified the aspidospermin (hypotensive) (Lyon et al., 1973), and yohimbine (alpha-adrenergic blocker) (Fumagali et al., 2008). Aspidosperma subincanum was the plant with the highest number of citations (19.2%) in ethnobotanical survey of medicinal plants used as hypocholesterolemic in central-western Brazil, being among the 10 most cited medicinal plants (Silva et al., 2010). Moreover, the bitter tonic of its bark is known by the population to stimulate circulatory functions (Federlin et al., 2014). In several regions of Brazil, Aspidosperma subincanum Mart., a species popularly known as “guatambú” is used for the treatment of cardiovascular diseases, including hypertension, hypercholesterolemia and erectile dysfunction (Campos et al., 2006; Tresvenzol et al., 2006). Its effects on cardiovascular functions have been scientifically explored by our research group (Bernardes et al., 2013), who demonstrated that in rats this species induces hypotension associated with vasodilation. It is known to contain indole alkaloids, some of which have been isolated (Kobayashi et al., 2002). The n-Hexane and dichloromethane fractions from Aspidosperma subincanum stem bark yielded two pure compounds, one of which has been identified as oleic acid, an unsaturated fatty acid, and the other a tetrahydropyridine alkaloid known as guatambuine (Santos et al., 2009). Despite the fact that folk medicine considers Aspidosperma subincanum an important adjuvant in cardiovascular diseases, its effects on the renal function have not yet been explored. Hence, this study set out to evaluate the acute diuretic effects of an orally administered extract obtained from the stem bark of Aspidosperma subincanum, and the mechanisms involved.

2. Material and methods 2.1. Plant material Bark from Aspidosperma subincanum Mart. was obtained from the Goiânia/GO region of Brazil in August 2010, and its identity was confirmed by Dr. Irani Fernandes Pereira Campos (Biological Sciences Institute/UFG). A voucher specimen was deposited in the herbarium of the Botany Department (UFG) and registered as number 21147. 2.2. Preparation of ethanol extract (EEAS) The botanical material was dried in a forced air oven at 40 1C for 72 h. It was then ground in a knife mill in the Pharmacognosy Laboratory at the Federal University of Goiás, and yielded 600 g of powdered plant material. A 95% ethanol solution was added to this material at a ratio of 1:3 (w/v). The mixture was mechanically stirred for 4 h and subsequently filtered through quantitative filter paper. The dynamic maceration process was repeated three times in a row. The solution extracted was subjected to evaporation under vacuum at a temperature below 40 1C until the solvent evaporated and the ethanol extract of Aspidosperma subincanum

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Mart. (EEAS) was obtained. It was stored at  20 1C, and exposure to light was minimized. EEAS was dissolved in saline just before administration, and given in doses of 60 and 120 mg/kg bw in a volume of 5 mL/kg bw. The oral doses employed in this study were based in previous study by Santos et al. (2009), which have shown that oral administration of doses up to 300 mg/kg of EEAS did not lead to any sign of toxicity in mice. 2.3. Animals Female Wistar rats (170–210 g) from the Central Animal House, Federal University of Goiás, were used for the experiments. The animals were housed in a temperature and light-controlled room (22 72 1C; 12 h light/dark cycle), and acclimatized in our laboratory for a period of at least one week before starting the experiment, with free access to water and food. The rats were handled in accordance with internationally accepted standard guidelines for the use of animals. All procedures were approved by the Animal Research Ethics Committee at the Federal University of Goiás, Goiânia, Brazil, in accordance with the internationally accepted principles for laboratory animal use and care, under protocol number 187/2011. 2.4. Drugs Furosemide (Lasixs 10 mg/mL, Sanofi Aventis Farmacêutica, São Paulo, Brazil), a high-ceiling loop diuretic, was used as the reference drug (positive control), and was dissolved in saline prior to administration. Piroxicam (Feldenes 20 mg/mL, Pfizer), a nonselective inhibitor of cyclooxygenase, was acquired to assess the action mechanism of EEAS. Saline (NaCl, 0.9%) was used as a control. 2.5. Pharmacological procedures 2.5.1. Acute diuretic activity Diuretic activity was determined according to the method described by Kau et al. (1984), but with some modifications. For these experiments, the rats were divided into four groups (n¼5 or 6) and fasted overnight for eighteen hours before testing, and with free access to tap water only. Before treatment (45 min), all animals received physiological saline (0.9% NaCl) in an oral dose of 1% body weight in order to impose a uniform water and salt load (Benjumea et al., 2005). Subsequently, two groups of rats were orally administered 5 mL/kg bw of the EEAS at 60 and 120 mg/kg of weight. Another group orally received 5 mL/kg bw of furosemide at 20 mg/kg. Control rats received the same amount of saline (5 mL/kg bw). Immediately after administration, the animals were placed in metabolic cages. Urine was collected in a graduated cylinder and its volume was measured and recorded 1, 2, 4, 6, 8, 12 and 24 h after treatment. Cumulative urine excretion was calculated in relation to body weight and expressed as mL/100 g. Electrolyte (Na þ and K þ ) concentration was estimated from each urine sample and expressed as mmol/L. And pH was verified from a pooled urine sample from each rat at the end of the experiment (24 h). The diuretic (volume treated group/volume control group) and saluretic indexes (mmol/L treated group/control group) were calculated. 2.5.2. Assessment of the role of prostaglandins in the diuretic effects of EEAS For these experiments four groups of rats were used according to the methodology previously described but with minor modifications (Gasparotto Jr. et al., 2009). Accordingly, eighteen hours before testing, the rats (n¼5 per group) were fasted overnight, with free access to tap water only. And in order to impose a uniform water and salt load, all

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animals received physiological saline (0.9% NaCl) in an oral dose of 1% body weight (Benjumea et al., 2005). The first group received the vehicle (0.9% NaCl) orally and was considered the control group; the second group was orally treated with EEAS 120 mg/kg p.o. only; the third group was pretreated with 5 mg/kg piroxicam (a cyclooxygenase inhibitor) administered intraperitoneally, 30 min before the single oral dose of the EEAS 120 mg/kg (EEAS 120þ Piroxicam). For comparative purposes, the fourth group received piroxicam 5 mg/kg and was subsequently treated with physiological saline (5 mL/kg bw) (Vehicleþ Piroxicam). Urine was collected in a graduated cylinder and its volume was measured and recorded 2, 4, and 8 h after treatment. Cumulative urine excretion was expressed as mL/100 g body weight. The concentrations of sodium and potassium were determined for each urine sample and expressed as mmol/L. In addition, pH was verified at the end of the experiments. 2.5.3. Analytical procedures Urinary levels of sodium and potassium were measured using a flame photometer (Analog Model 400, Corning Life Sciences, NY, USA). The instrument was calibrated with standard solutions containing different concentrations of Na þ and K þ . The electrolytes in EEAS in the same concentration used in the experiments were also measured. Physico-chemical parameters were analyzed in total fresh urine samples at the end of the experiments using a urine dipstick test (Urofitas Prodimol, Belo Horizonte, Brazil), and the pH was determined using a pH-meter (model pHb 500, Ionlab produtos laboratoriais, Curitiba, PR, Brazil). 2.6. Statistical analysis Results are expressed as the mean 7standard error of the mean (S.E.M.) of five animals in each group. Statistical significance was determined through the One-Way Analysis of Variances (ANOVA), followed by the Newman–Keuls test. A p-value of less than 0.05

was considered statistically significant. Graphs were drawn and statistical analysis was carried out using GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego, CA, USA).

3. Results 3.1. Diuretic, natriuretic and kaliuretic effects of EEAS Oral administration of EEAS increased the urinary flow in a dose dependent manner (Fig. 1). EEAS 60 and 120 mg/kg significantly increased the urinary output from the first hour onwards and continuously throughout the whole study period. The cumulative urinary excretions at 24 h after treatment with EEAS 60 and 120 mg/kg were: 7.5 70.7 and 10.3 7 0.9 mL. These values were significantly higher (p o0.001) when compared to the control group (4.3 70.4 mL). Therefore, EEAS 60 and 120 mg/kg led to a relative increase in cumulative diuresis of around 77.07 8.7% and 142.9 78.9%, respectively, compared to the control group. Moreover, the cumulative urinary volume found in EEAS 120 mg/kgtreated animals after 24 h was not any different from the diuresis obtained in animals treated with furosemide, as noted in the diuretic index of 2.4 and 2.5 (Table 1). Equally important, the diuretic index of EEAS 60 mg/kg was 1.8. The quantity of the urinary electrolytes sodium and potassium was measured in all samples collected, and the results were summarized at 2, 4, 8, 12 and 24 h after treatment as shown in Table 2. Similar to furosemide-treated animals, samples of urine from animals treated with EEAS 60 and 120 mg/kg also presented higher concentrations of Na þ and K þ when compared to the control rats from the second hour onwards after treatment, and continuously throughout the study period. Hence, EEAS 120 mg/kg increased sodium excretion by around 104.6711.1% at 2 h after treatment when compared to control values (EEAS 120 mg/kg 2 h, 139.0715.5 mmol/L vs. control, 67.977.8 mmol/L). Similarly, the reference drug furosemide

Fig. 1. Diuretic effect of a single oral administration of the ethanol extract of Aspidosperma subincanum (EEAS) in rats. (A) Urinary output presented by animals treated with the EEAS 60 and 120 mg/kg. (B) Na þ and (C) K þ levels in urine after EEAS administration. Na þ and K þ were reported in the urine collected for 24 h. The results show the mean 7S.E.M. of five animals per group. np o 0.05; nnp o0.01; nnnp o 0.001 compared to control. #p o 0.01 compared to furosemide group in 24 h.

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Table 1 Effect of oral administration of the EEAS on urinary volume and electrolyte excretion (24 h). Group

n

Urine volume (mL/100 g/24 h)

Diuretic indexa

Na þ (mmol/L)

K þ (mmol/L)

Saluretic indexb Na þ K þ

Na þ /K þ ratio

Control Furosemide (20 mg/kg) EEAS (60 mg/kg) EEAS (120 mg/kg)

5 5 5 6

2.1 70.2 5.3 70.4nnn 3.8 70.3nnn 5.2 70.5nnn

– 2.5 1.8 2.4

739.17 49.9 1015.5 7 28.8nnn 1122.4 7 59.2nnn 1237.0 7 40.3nnn

544.9 7 15.1 982.6 7 35.6nnn 844.2 7 31.2nnn 866.97 20.1nnn

– 1.4 1.8 1.5 1.5 1.7 1.6

1.4 1.0 1.3 1.4

The results show the mean values and standard errors; n ¼number of pairs used in each group. nnn

a b

p o 0.001 in comparison with the control group (vehicle). Diuretic index¼ volume treated group/volume control group. Saluretic index ¼mmol/L treated group/mmol/L control group.

Table 2 Effect of oral administration of ethanol extract of EEAS on urinary electrolytes (Na þ and K þ ) at 2, 4, 8, 12 and 24 h after treatment. Cumulative values are reported as mean 7 SEM for five rats in each group. Group

Control Furosemide (20 mg/kg) EEAS (60 mg/kg) EEAS (120 mg/kg) Control Furosemide (20 mg/kg) EEAS (60 mg/kg) EEAS (120 mg/kg)

Na þ



nn

Na þ (mmol/L) and K þ (mmol/L) 2h

4h

8h

12 h

24 h

67.9 7 7.9 168.67 18.7nnn 96.17 15.0 139.0 7 15.5nn 51.4 7 4.6 79.5 7 6.5nnn 83.4 7 13.8nnn 93.6 7 9.5nnn

249.6 7 15.2 376.7 7 17.8nnn 418.17 18.4nnn 498.07 42.0nnn 206.07 11.0 314.0 7 6.8nnn 248.4 7 20.6nnn 256.0 7 14.7nnn

402.4 734.5 558.8 736.9nn 663.7 719.3nnn 790.4 718.9nnn 291.6 717.6 500.0 74.2nnn 480.6 730.2nnn 490.4 725.4nnn

538.9 745.5 748.5 724.3nnn 781.6 739.3nnn 1044.4 741.9nnn 393.0 722.7 716.0 740.0nnn 641.4 727.3nnn 753.9 722.3nnn

739.17 49.9 1015.0 7 28.8nnn 1122.4 7 59.2nnn 1237.0 7 40.3nnn 544.9 7 15.1 982.6 7 35.6nnn 844.2 7 31.2nnn 867.0 7 20.1nnn

p o0.01 compared to controls. p o 0.001 compared to controls.

nnn

significantly increased sodium excretion compared with the control group (furosemide 2 h, 168.6718.7 mmol/L). From 4th hour until the end of the experiment, the group treated with EEAS 120 mg/kg provided a greater excretion of Na þ than the furosemide group (about 18% more in 24 h; p o0.01), while the group treated with EEAS 60 mg/kg provided a similar excretion of Na þ when compared to the group that received furosemide, so there was no statistical difference between 12 and 24 h after administration (Table 2). Both concentrations of EEAS administered provided higher concentrations of K þ when compared with the amount of this electrolyte found in urine from control rats, during the 24 h of the experiment (EEAS 60 mg/kg 844.2731.2 mmol/L; EEAS 120 mg/kg 866.9720.0 mmol/L vs. control 544.9715.1 mmol/L). The furosemide group showed significantly higher amounts of K þ in the urine when compared with the other groups. On the other hand, the EEAS 60 and 120 mg/kg presented an interesting sparing effect mainly on K þ when compared to that potent diuretic, as noted in the saluretic index of 1.8 for furosemide, and 1.6 for EEAS 120 mg/kg, considering the electrolyte K þ (Table 1). No significant change in the urinary pH profile was observed after 24 h, following the EEAS or furosemide administration. Moreover, there was no alkalization of urine (pH: EEAS 120 mg/kg 7.170.2 vs. control 7.470.9). Sodium and potassium were also measured in the EEAS. The first electrolyte was not detected in the dose used in the experiments, while the potassium content was 16.370.3 mmol/L. And the other physico-chemical parameters analyzed in total fresh urine samples at the end of the experiments were normal according to the urine dipstick tests (data not shown). 3.2. Cyclooxygenase inhibition hinders the diuretic effect of the EEAS The oral administration of a single dose of EEAS 120 mg/kg in rats pre-treated with piroxicam (5 mg/kg) reduced diuresis during the experiment, by approximately 54.2 7 13.8% after 2 h, by

50.3 712.4% after 4 h and by 38.97 4.8% after 8 h, compared with the group that only received EEAS 120 mg/kg. Pre-treatment with piroxicam presented no effects in the control group (vehicle) (Fig. 2). Prior administration of piroxicam (5 mg/kg) completely inhibited the natriuretic and kaliuretic effects of EEAS 120 mg/kg in rats 4 h after the treatment. Urinary sodium excretion reduced by around 38.4 78.5% at 4 h and by around 39.0 76.7% at 8 h, after treatment, compared with the group that only received EEAS 120 mg/kg (Fig. 3). Urinary potassium excretion also reduced by around 28.2 76.2% at 4 h and 47.1 75.2% at 8 h after treatment, compared with the control group (Fig. 3).

4. Discussion and conclusions This study examined the diuretic effects in rats of ethanol extract from the stem bark of Aspidosperma subincanum. Oral administration was chosen as that is how it is used in folk medicine. The mechanism of action by which diuresis was induced was also investigated and compared with furosemide and the control group (vehicle). The acute treatment of rats with EEAS exerted a significant diuretic effect from the first hour when compared with the control group, however this effect was lower than that of furosemide for this first period. The EEAS 120 mg/kg dose was more efficient than the 60 mg/kg dose, with a diuretic index of 2.4 (volume treated group/volume control group), and was similar to the diuretic index of furosemide (2.5). Therefore, there was no statistical difference in urinary excretion at 24 h between the group treated with EEAS 120 mg/kg and that treated with furosemide. In the rats orally treated with EEAS, the diuresis levels were approximately 77.0% and 142.9% higher than the control group for the doses of 60 and 120 mg/kg, respectively, in the cumulative urine of 24 h. Hence, the EEAS induced a dose-dependent urinary excretion. On

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the other hand, the oral administration of furosemide induced a significant increase in diuresis for all intervals, with greater intensity at the beginning of the experiments. The time difference for the diuretic action of these substances is related to the characteristics of the gastrointestinal absorption of the extract components, while furosemide acts quickly as a result of its bioavailability (Novaes et al., 2013). In addition, other pharmacokinetic considerations could be related to the duration of the diuretic effect as possible active metabolites in the extract or active components that have a long half-life, or even the prolonged action at the site (Holford and Sheiner, 2011). As for the electrolyte excretion provoked by EEAS, we observed a significant increase in the urinary excretion of Na þ and K þ in the two doses used in our experiments, although the potassium

Fig. 2. Inhibitory effect of piroxicam on urinary volume induced by the ethanol extract of Aspidosperma subincanum in rats. Groups of rats received either the EEAS 120 mg/kg, or vehicle (NaCl, 0.9%), with or without previous administration of piroxicam (5 mg/kg, i.p.). The results show the mean 7 S.E.M. Statistical analyses were performed by means of One-Way Analysis of Variances (ANOVA) followed by the Newman–Keuls test. nnp o0.01 and nnnp o 0.001 when compared with the control group. #po 0,001 when compared with the effect of the EEAS without piroxicam.

excretion with EEAS 120 mg/kg at 24 h after treatment was around 11.872.3% less than that seen in the furosemide group, which indicates an interesting sparing effect mainly on K þ when compared with furosemide. The diuretic activity of orally administered EEAS was evaluated in normal rats, after a single dose, and was found to be similar or more effective when compared with furosemide, a high-ceiling loop diuretic. Thus, our findings suggest that EEAS could have some action on the loop of Henle, or at least, to present the same effect when compared to the loop diuretic. In addition, this diuretic effect does not seem to be related to K þ extract content, and the urinary pH remained mostly unchanged throughout the study for both concentrations of EEAS. Therefore, all the evidence in this study corroborates the ethnopharmacological application of Aspidosperma subincanum in cardiovascular diseases. Several plants have been used in Brazilian folk medicine to induce a diuretic effect, but there is little published data to support this ethnopharmacological indication. Gasparotto Jr. et al. (2009) found that diuresis values were approximately 72% and 120% for doses of 150 and 300 mg/kg of hydroethanolic extract of Tropaeolum majus, respectively. However, there was a significant increase in sodium excretion only for the group treated with 300 mg/kg. On the other hand, the highest dose of hydroethanolic extract of Achillea millefolium increased diuresis by around 30–60% between 4 and 8 h after administration when compared to the control values, and was accompanied by increased urinary excretion of Na þ and K þ (Souza et al., 2013). The renal effect of the extracts could result from the decrease in reabsorption of electrolytes and water, which could come about by modulating the flow of filtration and/or from a tubular effect (Sadki et al., 2010). Na þ and water reabsorption are tightly regulated by both nonhormonal and hormonal factors, including the renal–angiotensin–aldosterone system and prostaglandins (Chi et al., 2008). The major prostaglandin produced in the kidney, Prostaglandin E2 (PGE2), promotes sodium excretion via inhibition of sodium transport in the distal nephron. In fact, dietary salt loading induces the expression of cyclooxygenases in renal medullary interstitium, binds to EP1 and/or EP3 receptors on the

Fig. 3. Inhibitory effect of piroxicam on electrolyte excretion induced by the EEAS 120 mg/kg in rats. Groups of rats received either the EEAS (120 mg/kg), or vehicle (NaCl 0.9%), with or without previous administration of piroxicam (5 mg/kg). (A), (B) and (C) Na þ levels in the urine collected at 2 h, 4 h and 8 h, respectively. (D), (E), and (F) K þ levels in the urine collected at 2 h, 4 h and 8 h, respectively. The results show the mean 7 S.E.M. Statistical analyses were performed by means of One-Way Analysis of Variances (ANOVA) followed by the Newman–Keuls test. np o0.05 and nnnp o 0.001 when compared with the control group. #p o 0,001 when compared with the effect of the EEAS without piroxicam.

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basolateral membrane of the collecting duct, and inhibits Na þ absorption (Chi et al., 2008; Yuhki et al., 2011). In this study, EEAS showed a greater natriuretic than kaliuretic effect. The Na þ /K þ ratio could define the nature of the diuretic mechanism. The Na þ /K þ ratio for furosemide is approximately 1, which means that it eliminates the two electrolytes equally. On the other hand, with EEAS 60 and 120 mg/kg this ratio was greater than 1 (1.33 and 1.43, respectively), with a lower excretion of K þ than Na þ , which could cause fewer side effects than drugs now available. It is worth noting that both COX-1 and COX-2 have been found in different regions and cells in the kidneys, including human kidneys, and are involved in renoprotection and regulation of electrolyte excretion/reabsorption under both physiological and pathological conditions (Vio et al., 1997; Green et al., 2012). When rats were treated with EEAS along with piroxicam, a nonselective inhibitor of cyclooxygenase, there was a 38.9% reduction in the diuretic ability of EEAS after 8 h, which would suggest that the effect of one or more active compounds of this extract partially facilitates the release of prostaglandins. Some herbs induce diuresis by stimulating the thirst center in the hypothalamus and thereby enhancing fluid intake. Some plants induce diuresis because of their high salt content. It is well known that potassium overloading produces urinary excretion of the osmotic type and this occurs when the kidney tubules are incapable of absorbing it (Durairaj et al., 2007; Martín-Herrera et al., 2008). The abovementioned data lead to the hypothesis that the diuretic effect of EEAS is not related to an osmotic-type mechanism. Quantitative determinations of the ions present in the EEAS show the presence of extremely low amounts of potassium, which suggests that its diuretic effect is not due to its high potassium content. Flavonoids, saponins and organic acids have shown a diuretic effect, and several isoflavonoids have been reported to cause inhibition of the Na þ –K þ –2Cl  cotransporter, as well as an increase in natriuresis and kaluresis (Rodríguez, et al., 2013). Furthermore, the phytochemical analysis of the stem bark of Aspidosperma subincanum revealed the presence of indole alkaloids, saponins, terpenoids, steroids and tannins (Kobayashi et al., 2002; Santos et al., 2009). The possibility of finding a medicinal plant with diuretic effects similar to those of useful synthetic drugs but with fewer side effects seems very promising because an earlier study has reported hypotensive and vasodilatory effects when the EEAS was administered intravenously in rats (Bernardes et al., 2013). And in addition, the oral administration of doses up to 300 mg/kg of EEAS did not lead to any sign of toxicity in mice (Santos et al., 2009). In conclusion, this study demonstrated that the extract obtained from the stem bark of Aspidosperma subincanum Mart. increases diuresis, natriuresis and kaliuresis when orally administered to rats, supporting the ethnopharmacologic indication in cardiovascular diseases. This action appears to involve prostaglandins with a consequent increase in the glomerular filtration rate and natriuresis. But further studies would be required to evaluate its long-term effects on diuresis, and to identify the compounds responsible for such effects.

Acknowledgments This work received Grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil), and by Fundação de Amparo à Pesquisa do Estado de Goiás (FAPEG, Brazil). References Barbosa, W.L.R., Tavares, I.C.C., Soares, D.C., 2003. Alcaloides de Aspidosperma auriculatum Standl. Revista Brasileira de Farmacognosia 13, 6–8.

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